海洋菌株Aeromonas sp.YS-54来源几丁质酶的制备与性质研究

Preparation and Characterization of Chitinase from Marine Bacteria Aeromonas sp.YS-54

为实现甲壳类原料的高效降解与N-乙酰氨基寡糖的绿色制备,本研究以青岛近海土壤中筛选的菌株YS-54为材料,使用60%硫酸铵沉淀、Sephadex G-100凝胶过滤层析制备几丁质酶;对几丁质酶的最适温度、最适pH、底物偏好性等性质进行表征;使用质谱技术确认了酶解产物的聚合度。结果表明,菌株YS-54为Aeromonas属,该菌株来源几丁质酶比活力为23.44 U/mg,分子量为63与75 kDa。几丁质酶在40℃和pH为5.0条件下具有良好的稳定性,Ba^(2+)、Co^(2+)、Mg^(2+)与TritonX-100显著促进了几丁质酶活力(P<0.05),而Fe^(3+)、Cu^(2+)、SDS对酶活力起抑制作用。几丁质酶对α-几丁质的比活力为7.99 U/mg,为水解胶体几丁质比活力的34.09%。几丁质酶对胶体几丁质与α-几丁质的酶解产物聚合度分别为1~4与1~3。Aeromonas sp.YS-54来源几丁质酶在较宽的温度与pH范围内保持稳定,同时对α-几丁质具有良好的水解能力,可为甲壳类废弃物的高效综合利用提供技术支持。

To achieve efficient degradation of crustacean raw materials and eco-preparation of N-acetyl-oligosaccharides,YS-54 strain screened from offshore soil in Qingdao was used for chitinase preparation in the present study.Chitinase was prepared through 60%ammonium sulfate precipitation and Sephadex G-100 gel filtration chromatography.Enzymatic properties such as optimal temperature,optimal pH,and substrate specificity were characterized.Mass spectrometry was used to determine the polymerization degree of enzymatic products.The 16S rRNA identification result showed that YS-54 strain belonged to the Aeromonas species.Chitinase from Aeromonas sp.YS-54 showed a specific activity of 23.44 U/mg.The molecular weights of chitinase were 63 and 75 kDa as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Chitinase was stable under the condition of 40℃and pH5.0.The enzymatic activity was significantly activated by Ba^(2+),Co^(2+),Mg^(2+)and TritonX-100(P<0.05),while inhibited by Fe^(3+),Cu^(2+)and SDS.The specific activity of chitinase towardα-chitin was 7.99 U/mg,34.09%of that toward colloidal chitin.The polymerization degree of enzymatic hydrolysis products from colloidal chitin andα-chitin were 1~4 and 1~3,respectively.Chitinase from Aeromonas sp.YS-54 was stable in broad range of temperature and pH,meanwhile showed high catalytic efficiency towardα-chitin.The hydrolysis characteristics of chitinase from Aeromonas sp.YS-54 would provide technical support for the comprehensive utilization of crustacean waste.